Recent technological advances in the biomedical sciences have made it possible to take inventory of the components of biological systems at an unprecedented level of detail. This applies not only to genes and genetic regulatory elements, but also to the proteins and small molecules that exist within living organisms. Critical to understanding the functional interplay of these components, in both health and disease, is the quantitative measurement of their concentration in biological samples. For genes and their transcription products this is readily accomplished through techniques such as the quantitative polymerase chain reaction (qPCR), which have revolutionized molecular biology and clinical diagnostics. For proteins and small molecules, however, the analytical methods are more specialized, and do not benefit from exponential signal amplification as occurs with qPCR. During the previous project period, a new approach was developed for the quantitative detection of proteins and small molecules that has high specificity and achieves high sensitivity due to exponential signal amplification. The approach relies on a novel class of self-replicating nucleic acid enzymes that undergo exponential amplification at constant temperature, and can be made to do so contingent upon recognition of a target ligand. The proposed research will expand the generality of the system so that it can be applied to a broad range of targets, including disease-related proteins, drugs, and metabolites. A real-time fluorescent assay will be developed that enables high-throughput, multiplex analysis using standard instrumentation. The self-replicating enzymes will be optimized using a combination of site-specific chemical modification and directed evolution so that they are stable in biological samples and operate with high catalytic efficiency. The method for linking the ligand-recognition element to the replicating enzymes will be generalized and applied to a variety of proteins of relevance to biosensing and clinical diagnostics. The proposed research also will investigate a potentially more far-reaching approach that takes advantage of the unique ability of the replicating enzymes to evolve in a self-sustained manner, enabling them to configure themselves to recognize a target molecule. This is analogous to the maturation of antigen recognition by the immune system, but would occur entirely in the test tube within the context of a synthetic genetic system. These efforts will provide the opportunity to develop a new class of smart materials to help keep pace with the rapid rate of discovery of novel biological targets.